In cochlear inner hair cells (IHCs), Cav1.3 L-type voltage-gated Ca2+ channels mediate Ca2+ action potentials before the onset of hearing and Ca2+ signals that trigger exocytosis of neurotransmitter from IHCs onto auditory nerve afferents. These functions of Cav1.3 are crucial for the development and maintenance of hearing: mice lacking Cav1.3 are congenitally deaf, as are mice with upregulated Cav1.3 channels due to thyroid hormone deficiency. Thus, factors that regulate these channels can profoundly impact this first synapse in the auditory pathway. In this proposal, we will characterize two factors we have found to regulate Cav1.3 channels in IHCs: (1) CaBPs, which are a family of calmodulin-like Ca2+-binding proteins and (3) harmonin, a protein that corresponds to a genetic locus of Usher syndrome, a leading cause of combined deafness and blindness in humans. We propose that the macromolecular assembly of Cav1.3 with proteins such as harmonin and CaBPs dictates the strength and localization of Ca2+ signals in IHCs, and is crucial for the development and maintenance of auditory transmission. The goal of this proposal is to characterize the molecular mechanisms and functional consequences of these Cav1.3 interactions, and their physiological significance for hearing. Accomplishing this objective will clarify the modulatory influences of auditory Cav1.3 channels, which may be targeted pharmacologically in novel strategies to offset pathological changes involved in hereditary forms of deafness. The proposed research will modulate voltage-gated Ca2+ channels in auditory hair cells. We will elucidate new structure/function relationships and modulatory mechanisms, which may be altered in hereditary forms of deafness.